Compositions and methods for treating alzheimer&#39;s disease

ABSTRACT

The present invention provides compositions for reducing amyloid plaque burden associated with Alzheimer&#39;s disease and methods of using the same.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.14/191,984, filed on Feb. 27, 2014, which is a continuation of U.S.application Ser. No. 13/467,519, filed on May 9, 2012 (issued as U.S.Pat. No. 8,697,627 on Apr. 15, 2014), which claims the benefit of U.S.Provisional Application No. 61/483,919, filed May 9, 2011, each of whichare hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Alzheimer's disease is the most common cause of dementia, and ischaracterized by the loss of intellectual and social abilities severeenough to interfere with daily functioning. In Alzheimer's disease,healthy brain tissue degenerates, causing a steady decline in memory andmental abilities. Alzheimer's disease is not a part of normal aging, butthe risk of the disorder increases with age. About 5 percent of peoplebetween the ages of 65 and 74 have Alzheimer's disease, while nearlyhalf the people over the age of 85 have Alzheimer's.

Two types of neuron pathology, plaques and tangles, are common inpatients with Alzheimer's disease. Extracellular plaques are clumps of anormally harmless protein called beta-amyloid (Aβ) which may interferewith communication between brain cells. Tangles are the internal supportstructure for brain cells depends on the normal functioning of a proteincalled tau. In people affected with Alzheimer's disease, threads of tauprotein undergo alterations that cause them to become twisted. Manyresearchers believe this may seriously damage neurons, causing them todie and leading to memory deficit.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B summarize the effects of VX-745 on the area percentageof amyloid plaques in the cortex (FIG. 1A) and hippocampus (FIG. 1B)following two-week administration of VX-745 (3 mg/kg BID).

FIG. 2 summarizes the effects of VX-745 on IL-1β as compared towild-type and vehicle controls.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION Definitions

Carrier: The term “carrier” refers to any chemical entity that can beincorporated into a composition containing an active agent (e.g., a p38inhibitor) without significantly interfering with the stability and/oractivity of the agent (e.g., with a biological activity of the agent).In certain embodiments, the term “carrier” refers to a pharmaceuticallyacceptable carrier. An exemplary carrier herein is water.

Combination. As used herein, the term “combination,” “combined,” andrelated terms refers to a subject's simultaneous exposure to two or moretherapeutic agents in accordance with this invention. For example, anagent of the present invention (e.g., a p38 inhibitor) may beadministered with another therapeutic agent simultaneously orsequentially in separate unit dosage forms or together in a single unitdosage form. Accordingly, the present invention provides, among otherthings, dosing regimens that involve administering at least an agent ofthe present invention (e.g., a p38 inhibitor), an additional therapeuticagent, and a pharmaceutically acceptable carrier, adjuvant, or vehicle(the pharmaceutically acceptable carrier, adjuvant, or vehicle typicallybeing in association with one or both of the p38 inhibitor and theadditional therapeutic agent).

Formulation. The term “formulation” refers to a composition thatincludes at least one active agent (e.g., a p38 inhibitor) together withone or more carriers, excipients or other pharmaceutical additives foradministration to a patient. In general, particular carriers, excipientsand/or other pharmaceutical additives are selected in accordance withknowledge in the art to achieve a desired stability, release,distribution and/or activity of active agent(s) and which areappropriate for the particular route of administration.

Low dose. The term “low dose” as used herein refers to a dose that isbelow the therapeutically effective amount of the reference p38inhibitor when administered to treat a disease other than Alzheimer'sdisease. In some embodiments, the term “low dose” refers to a dose thatis one or more orders of magnitude lower than the therapeuticallyeffective amount of the reference p38 inhibitor when administered totreat a disease other than Alzheimer's disease. In some embodiments, theterm “low dose” refers to a dose that is one-half, one-third,one-fourth, one-fifth, one-sixth, one-seventh, one-eighth or less thanthe therapeutically effective amount of the reference p38 inhibitor whenadministered to treat a disease other than Alzheimer's disease. Forexample, a therapeutically effective unit dose of VX-745 for thetreatment of rheumatoid arthritis in humans is 250 mg. In someembodiments, a “low dose” of VX-745 is within the range of about 1 mg toabout 100 mg. In some embodiments, a “low dose” of VX-745 is within therange of about 1 mg to about 50 mg. In some embodiments, a “low dose” ofVX-745 is within the range of about 1 mg to about 30 mg. In someembodiments, a “low dose” of VX-745 is within the range of about 1 mg toabout 10 mg. In some embodiments, a “low dose” of VX-745 is within therange of about 1 mg to about 5 mg. In some embodiments, a “low dose” ofVX-745 is about 3 mg. In some embodiments, a “low dose” of VX-745 iswithin the range of 5-10 mg. In some embodiments, a “low dose” of VX-745is within the range of 10-20 mg. In some embodiments, a “low dose” ofVX-745 is within the range of 20-30 mg.

Neuroimaging. As used herein, the term “neuroimaging” refers to atechnique which directly or indirectly images the structure or functionof the brain. In some embodiments, the term “neuroimaging” refers to atechnique selected from computerized axial tomography (CAT or CT),single photon emission computed tomography (SPECT), positron emissiontomography (PET), magnetic resonance imaging (MRI) or functionalmagnetic resonance imaging (fMRI). In some embodiments, a neuroimagingtechnique employs one or more imaging agents such as radioactive,fluorescent or other detectable ligands. In some embodiments, afluorescent ligand is Pittsburgh compound B([N-Methyl-¹¹C]-(4′-methylaminophenyl)-6-hydroxybenzothiazole), afluorescent analog of thioflavin T. In some embodiments, a radioactiveligand is Amyvid® (florbetapir F18) or 18F-flutemetamol. In someembodiments, the neuroimaging technique is PET scan using Pittsburghcompound B as an imaging agent. In some embodiments, the neuroimagingtechnique is PET scan using Amyvid® as an imaging agent. In someembodiments, the neuroimaging technique is PET scan using18F-flutemetamol as an imaging agent.

Neuroimage. As used herein, the term “neuroimage” refers to an image orpicture generated by a neuroimaging technique. In some embodiments, a“neuroimage” refers to one or more of CAT (or CT), SPECT, PET, MRI orfMRI scans.

Parenteral. The term “parenteral” as used herein includes subcutaneous,intravenous, intramuscular, intra-articular, intra-synovial,intrasternal, intrathecal, intrahepatic, intralesional and intracranialinjection or infusion techniques. Preferably, the compositions areadministered orally, intraperitoneally or intravenously. Sterileinjectable forms of the compositions of this invention may be aqueous oroleaginous suspension. These suspensions may be formulated according totechniques known in the art using suitable dispersing or wetting agentsand suspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium.

Patient. The term “patient”, as used herein, means a mammal to which aformulation or composition comprising a formulation is administered, andin some embodiments includes humans.

Pharmaceutically acceptable carrier, adjuvant, or vehicle. The term“pharmaceutically acceptable carrier, adjuvant, or vehicle” refers to anon-toxic carrier, adjuvant, or vehicle that does not destroy thepharmacological activity of the compound with which it is formulated.Pharmaceutically acceptable carriers, adjuvants or vehicles that may beused in the compositions of this invention include, but are not limitedto, ion exchangers, alumina, aluminum stearate, lecithin, serumproteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

Selective p38 Inhibitor. As used herein, the phrase “selective p38inhibitor” refers to an agent which elicits a biological effect (i.e.,an inhibitory or antagonistic effect) on p38 mitogen-activated proteinkinase (also referred to as p38 MAPK) that is at least one order ofmagnitude greater than another kinase. For example, in some embodiments,a selective p38 inhibitor is an inhibitor which is selective for p38MAPK over other protein kinases or tyrosine kinases. In someembodiments, a selective p38 inhibitor is selective for one p38 MAPKisoform over another. For example, in some embodiments, a selective p38inhibitor refers to an inhibitor that has greater antagonistic effectagainst one of alpha (α), beta (β), gamma (γ) or delta (δ) p38 MAPKisoforms over another isoform. In some embodiments, a selective p38inhibitor refers to an inhibitor that has greater antagonistic effectagainst the p38a isoform of MPAK as compared to the p38β, p38γ and/orp38δ isoforms. Representative selective p38 inhibitors include, but arenot limited to, RWJ 67657, SCIO 469, EO 1428, Org 48762-0, SD 169, SB203580, SB 202190, SB 239063, SB 220025, VX 745, SB 242235, VX 702,SD-282, PH-797804 and others.

Therapeutic agent. As used herein, the phrase “therapeutic agent” refersto any agent that elicits a desired biological or pharmacological effectwhen administered to an organism.

Therapeutically effective amount and effective amount. As used herein,and unless otherwise specified, the terms “therapeutically effectiveamount” and “effective amount” of an agent refer to an amount sufficientto provide a therapeutic benefit in the treatment, prevention and/ormanagement of a disease, disorder, or condition, e.g., to delay onset ofor minimize (e.g., reduce the incidence and/or magnitude of) one or moresymptoms associated with the disease, disorder or condition to betreated. In some embodiments, a composition may be said to contain a“therapeutically effective amount” of an agent if it contains an amountthat is effective when administered as a single dose within the contextof a therapeutic regimen. In some embodiments, a therapeuticallyeffective amount is an amount that, when administered as part of adosing regimen, is statistically likely to delay onset of or minimize(reduce the incidence and/or magnitude of) one or more symptoms or sideeffects of a disease, disorder or condition. In some embodiments, a“therapeutically effective amount” is an amount that enhancestherapeutic efficacy of another agent with which the composition isadministered in combination. In some embodiments, a therapeuticallyeffective amount for administration to a human corresponds to areference amount (e.g., a therapeutically effective amount in an animalmodel such as a mouse model) adjusted for body surface area of a humanas compared with body surface area of the animal model, as is known inthe art (see, for example Reagan-Shaw et al., “Dose translation fromanimal to human studies revisited,” The FASEB Journal 22: 659-661(2007), the entirety of which is herein incorporated by reference). Insome embodiments, the reference therapeutically effective amount is anamount that is therapeutically effective in a mouse model, for example,as described herein. In some embodiments, the reference therapeuticallyeffective amount is within the range of about 0.0001 mg/kg to about 500mg/kg. In some embodiments, the reference therapeutically effectiveamount is within the range of about 0.0001 mg/kg to about 0.001 mg/kg.In some embodiments, the reference therapeutically effective amount iswithin the range of about 0.001 mg/kg to about 0.01 mg/kg. In someembodiments, the reference therapeutically effective amount is withinthe range of about 0.01 mg/kg to about 0.1 mg/kg. In some embodiments,the reference therapeutically effective amount is within the range ofabout 0.1 mg/kg to about 0.5 mg/kg. In some embodiments, the referencetherapeutically effective amount is within the range of about 0.5 mg/kgto about 1 mg/kg. In some embodiments, the reference therapeuticallyeffective amount is within the range of about 1 mg/kg to about 2.5mg/kg. In some embodiments, the reference therapeutically effectiveamount is within the range of about 2.5 mg/kg to about 10 mg/kg. In someembodiments, the reference therapeutically effective amount is withinthe range of about 10 mg/kg to about 50 mg/kg. In some embodiments, thereference therapeutically effective amount is within the range of about50 mg/kg to about 100 mg/kg. In some embodiments, the referencetherapeutically effective amount is within the range of about 100 mg/kgto about 250 mg/kg. In some embodiments, the reference therapeuticallyeffective amount is within the range of about 250 mg/kg to about 500mg/kg.

Treat or Treating. The terms “treat” or “treating,” as used herein,refer to partially or completely alleviating, inhibiting, delaying onsetof, reducing the incidence of, yielding prophylaxis of, amelioratingand/or relieving a disorder, disease, or condition, or one or moresymptoms or manifestations of the disorder, disease or condition.

Unit Dose. The expression “unit dose” as used herein refers to aphysically discrete unit of a formulation appropriate for a subject tobe treated (e.g., for a single dose); each unit containing apredetermined quantity of an active agent selected to produce a desiredtherapeutic effect when administered according to a therapeutic regimen(it being understood that multiple doses may be required to achieve adesired or optimum effect), optionally together with a pharmaceuticallyacceptable carrier, which may be provided in a predetermined amount. Theunit dose may be, for example, a volume of liquid (e.g., an acceptablecarrier) containing a predetermined quantity of one or more therapeuticagents, a predetermined amount of one or more therapeutic agents insolid form, a sustained release formulation or drug delivery devicecontaining a predetermined amount of one or more therapeutic agents,etc. It will be appreciated that a unit dose may contain a variety ofcomponents in addition to the therapeutic agent(s). For example,acceptable carriers (e.g., pharmaceutically acceptable carriers),diluents, stabilizers, buffers, preservatives, etc., may be included asdescribed infra. It will be understood, however, that the total dailyusage of a formulation of the present invention will be decided by theattending physician within the scope of sound medical judgment. Thespecific effective dose level for any particular subject or organism maydepend upon a variety of factors including the disorder being treatedand the severity of the disorder; activity of specific active compoundemployed; specific composition employed; age, body weight, generalhealth, sex and diet of the subject; time of administration, and rate ofexcretion of the specific active compound employed; duration of thetreatment; drugs and/or additional therapies used in combination orcoincidental with specific compound(s) employed, and like factors wellknown in the medical arts. In some embodiments, a unit dose of a p38inhibitor is about 1 mg, 3 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg,35 mg, 40 mg, 45 mg or 50 mg.

Pathology of Alzheimer's Disease

Alzheimer's disease pathology is characterized by the deposition ofextracellular amyloid plaques in the brain parenchyma andneurofibrillary tangles within neurons.

The primary component of extracellular amyloid plaques found in thebrains of Alzheimer's disease patients is abnormally folded beta-amyloidprotein (Aβ), a 36- to 43-amino acid peptide produced by proteolysis ofamyloid precursor protein (APP) by enzymes known as secretases. APP isan integral membrane protein expressed in many tissues and concentratedin the synapses of neurons. Its primary function is not known, though ithas been implicated as a regulator of synapse formation, neuralplasticity and iron export. The most common isoforms of Aβ are Aβ₄₀ andAβ₄₂; the shorter form is typically produced by cleavage that occurs inthe endoplasmic reticulum, while the longer form is produced by cleavagein the trans-Golgi network. The Aβ₄₀ form is the more common of the two,but Aβ₄₂ is the more fibrillogenic and is thus associated with diseasestates. Mutations in APP associated with early-onset Alzheimer's havebeen noted to increase the relative production of Aβ₄₂, and thus onesuggested avenue of Alzheimer's therapy involves modulating the activityof β- and γ-secretases to produce mainly Aβ₄₀.

In contrast, neurofibrillary tangles are intracellular aggregates ofmicrotubule-associated protein tau (MAPT). Tau proteins, abundant inneurons in the central nervous system but less common elsewhere,stabilize microtubules. Hyperphosphorylated tau (hTau) associates withother threads of tau, eventually forming neurofibrillary tangles insidenerve cell bodies. When this occurs, the microtubules disintegrate,collapsing the neuron's transport system, resulting in malfunctions inbiochemical communication between neurons and, eventually, cell death.

Recent evidence suggests that neuroinflammatory processes alsocontribute to the pathophysiology of Alzheimer's disease. See, e.g.,Hull et al., “Pathways of Inflammatory Activation in Alzheimer'sDisease: Potential Targets for Disease Modifying Drugs,” Curr. Med.Chem. 2002, 9, 83-88, the entirety of which is incorporated herein byreference. Microglia, the resident inflammatory cells of the brain, arefound in a highly activated state in the Alzheimer's disease brain,including morphological alterations, proliferation, increased expressionof cell surface receptors, and secretion of inflammatory cytokines andchemokines Microglia fulfill numerous different tasks within the centralnervous system (CNS) related to both immune response and the maintenanceof homeostasis. The main role of microglia is phagocytosis, or theengulfing of various materials. Engulfed materials include damagedneurons, plaques, cellular debris and infectious agents such as virusesand bacteria. Microglia accumulate at the site of newly formed Aβdeposits in the Alzheimer's disease brain and may help restrict plaquegrowth by degrading Aβ.

Recent studies have also shown that overexpression of IL-1β, aninflammatory cytokine, leads to reduced Aβ pathology in mouse models ofAlzheimer's disease. However, chronically activated microglia are alsoassociated with inflammatory cytokines including TNFα that cansubstantially block the ability of the microglia to remove or degradeAβ. Thus, the role of microglia in the pathophysiology of Alzheimer'sdisease is complex, with microglial activation exerting either abeneficial or detrimental effect depending on local conditions.

One signaling pathway through which neurons and microglia communicate isfractalkine (CX3CL1) and its cognate receptor (CX3CR1), a unique,one-to-one ligand-receptor chemokine pair. CX3CL1-CX3CR1 signaling hasbeen demonstrated to play an important role in neuroinflammation andneuroprotection. Notably, CX3CL1 is highly expressed in neurons whileCX3CR1 is exclusively expressed in microglia. One recent studydemonstrated that the inhibition or deletion of the microglial receptorCX3CR1 leads to an amelioration of the amyloid pathology in both rapidonset and gradual onset transgenic mouse models of Alzheimer's disease.See Lee et al., “CX3CR1 Deficiency Alters Microglial Activation andReduces Beat-Amyloid Deposition in Two Alzheimer's Disease MouseModels,” The American Journal of Pathology, 177(5): 2549-2562 (2010),the entirety of which is incorporated herein by reference. In fact,CX3CR1-deficient mice exhibited a dose-dependent reduction in Aβdeposition in the APPPS1 mouse model of Alzheimer's disease, suggestingthat CX3CR1 deficiency harnesses the beneficial effects of microglialactivation in response to Aβ. Moreover, the number of plaque-associatedmicroglia were decreased in the knockout mice as compared to control.However, despite the reduction in the number of microglia around the Aβdeposits in the CX3CR1-deficient animals, there was observed asignificant reduction in Aβ deposition, consistent with an enhancedcapacity of microglia to remove Aβ. Thus, CX3CR1 signaling appears toinhibit microglial phagocytosis and prevent effective Aβ clearance.These results suggest that alterations in CX3CL1-CX3CR1 signaling canlead to altered phagocytic capabilities of microglia.

Microglial neuroinflammation also promotes MAPT phosphorylation andaggregation through the overexpression of IL-1. Recently, CX3CR1deficiency has been shown to result in both enhanced microglialactivation and MAPT phosphorylation/aggregation in humanized tau mice.Researchers observed that transgenic humanized tau mice first develophyperphosphorylated MAPT at 3 months of age, MAPT aggregates at 9 monthsof age, and neuronal loss by 15 months of age. Significantly, by 12months of age, humanized tau mice exhibited microglia in the hippocampuswith shorter processes and rounder cell bodies consistent withmicroglial activation.

Without wishing to be bound by any particular theory, it is believedthat the activation of microglia in response to such inflammatorysignals, particularly neuroinflammatory processes, delays theaccumulation of beta amyloid plaques associates with Alzheimer'sdisease. Thus, inhibition or suppression of inflammatory cascades islikely to prevent microglial activation, leading to an increase inaccumulation of beta amyloid plaques and the progression of Alzheimer'sdisease.

p38 MAPK Inhibitors

Many extracellular stimuli, including pro-inflammatory cytokines andother inflammatory mediators, elicit specific cellular responses throughthe activation of mitogen-activated protein kinase (MAPK) signalingpathways. MAPKs are proline-targeted serine-threonine kinases thattransduce environmental stimuli to the nucleus. Once activated, MAPKsactivate other kinases or nuclear proteins through phosphorylation,including potential transcription factors and substrates. The novelmammalian reactivating protein kinase (p38/RK) MAPKs arestress-activated protein kinases that mediate responses to cellularstresses and inflammatory signals.

p38 MAPK activation occurs in the very early stages of Alzheimer'sdisease and is an important contributor to the inflammation of thebrain. See, e.g., Bhasker et al., “Regulation of Tau Pathology by theMicroglial Fractalkine Receptor,” Neuron 68:19-31 (2010), the entiretyof which is incorporated herein by reference. In fact, beta-amyloidfibrils in microglia stimulate rapid, transient activation of p38 MAPKresulting in inflammatory gene expression and upregulation ofproinflammatory cytokines Thus, activation of the p38 MAPK pathwayattenuates plaque accumulation and stimulates microglial plaquedegradation.

Moreover, researchers confirmed that enhancement of MAPT phosphorylationcould be blocked by preincubating neurons in vitro with a specific MAPKinhibitor, SB203580, indicating that the enhancement of MAPTphosphorylation occurred via a p38 MAPK-dependent pathway. Thus,research demonstrates that the role of p38 MAPK in Alzheimer's diseaseis complex, as it both stimulates microglial degradation of Aβ plaqueswhile simultaneously promoting MAPT phosphorylation, a process which canlead to neurofibrillary tangles and loss of neuronal function. See,e.g., Munoz, et al., “Targeting p38 MAPK pathway for the treatment ofAlzheimer's disease,” Neuropharmacology, 58(3):561-568 (2010),incorporated herein by reference in its entirety.

The role of p38 MAPK in the various stages of inflammation has promptedthe discovery of several compounds capable of inhibiting p38 (SB203580,RWJ 67657, L-167307, VX-745, RPR200765A and others). See, e.g., Kumar etal., “p38 MAP Kinases: Key Signaling Molecules as Therapeutic Targetsfor Inflammatory Diseases,” Nature Reviews, 2:717-726 (2003); Brown etal., “p38 MAP kinase inhibitors as potential therapeutics for thetreatment of joint degeneration and pain associated withosteoarthritis,” J. Inflammation 5:22 (2008), the entirety of each ofwhich is incorporated herein by reference. These pharmacologicalinhibitors are cytokine-suppressive anti-inflammatory drugs responsiblefor in vitro and in vivo inhibition of lipopolysaccharide-induced tumornecrosis factor-α (TNF-α) expression. Although p38 MAPK inhibitors havelong piqued the interest of Alzheimer's disease researchers, thecomplexity of the disease has limited the use of such agents. Moreparticularly, while p38 inhibitors block tau phosphorylation, theresulting decrease in the inflammatory cascades are expected to increaseAβ plaque accumulation due to the lack of microglial activation.

It has now been surprisingly found that p38 MAPK inhibitors reduceamyloid plaque burden within the central nervous system (CNS).Accordingly, the present invention encompasses the recognition that p38MAPK inhibitors are effective for reducing amyloid plaque burdenassociated with Alzheimer's disease. In some embodiments, the presentinvention provides a method of reducing amyloid plaque burden within thecentral nervous system (CNS). In some embodiments, the present inventionprovides a method of reducing amyloid plaque burden associated withAlzheimer's disease comprising administering to a patient in needthereof a p38 MAPK inhibitor.

Exemplary p38 MAPK Inhibitors

As generally described above, there has been extensive research directedtowards the discovery of p38 MAPK inhibitors for the treatment of thevarious stages of inflammation.

Exemplary p38 MAPK inhibitors can be found, for example, in Mayer etal., “p38 MAP kinase inhibitors: A future therapy for inflammatorydiseases,” Drug Discovery Today: Therapeutic Strategies 3(1): 49-54(2006); and Regan et al., “Pyrazole Urea-Based Inhibitors of p38 MAPKinase: from Lead Compound to Clinical Candidate,” J. Med. Chem. 2002,45, 2994-3008, the entirety of each of which are incorporated herein byreference. Table 1 lists representative p38 MAPK inhibitors.

TABLE 1

VX-702

BIRB 796 (Doramapimod)

TAK-715

SCIO 469

RWJ 67657

SB-681323

SB-242235

VX-745

SB-203580

L-167307

RPR-203494

RPR-200765A

PD 169316

SB-200025

JX 401

CMPD1

SKF 86002

SX 011

SD 282

EO 1428

SD 169

SB 220025

SB 202190

SB 239063

Org 48762-0

LY2228820

Vinorelbine

PH-797804

Asiatic acid

4-(4-(4-fluorophenyl)-1-(piperidin-4- yl)-1H-imidazol-5-yl)pyridine

(R)-N-(1-phenylethyl)-4-(6-(piperidin-4-yl)-3-(3-(trifluoromethyl)phenyl) pyridazin-4-yl)pyrimidin-2-amine

2-(4-fluorophenyl)-6-methoxy-3- (pyridin-4-yl)-1H-indole

3-(4-fluorophenyl)-2-(pyridin-4-yl)-1H- pyrrolo[3,2-b]pyridine

6-(2-(2,6-difluorophenyl)-4-phenyl-1H-imidazol-5-yl)-1-(isopropylsulfonyl)- 1H-benzo[d]imidazol-2-amine

4-(4-fluorophenyl)-5-(1-isopropyl-1H-benzo[d][1,2,3]triazol-6-yl)oxazole

(2-chloro-4-(4-fluoro-2- methylphenylamino)phenyl)(o- tolyl)methanone

5-(2,6-dichlorophenyl)-2-(2,4- difluorophenylthio)-6H-pyrimido[1,6-b]pyridazin-6-one

6-(1,4-diazepan-1-yl)-N²-(((1S,2R,5S)-6,6-dimethylbicyclo[3.1.1]heptan-2- yl)methyl)-N4-phenyl-1,3,5-triazine-2,4-diamine

1-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5- yl)-3-p-tolylurea

VX-745 is a selective small-molecule inhibitor of p38 MAPK developed byVertex Pharmaceuticals for the treatment of rheumatoid arthritis (RA).The inhibition of MAPK by VX-745 blocks the downstream synthesis ofinflammatory cytokines TNF-α, IL-1β and IL-6. Because VX-745 exhibitedsignificant anti-inflammatory activity in rodent arthritis models,Vertex initiated a clinical trial in human rheumatoid arthritis (RA).However, patients treated with 250 mg VX-745 b.i.d. experienced adverseevents, including gastrointestinal effects such as diarrhea andabdominal pain, and elevations in liver transaminases. Moreover, VX-745is known to penetrate the blood brain barrier (BBB) in animals. In fact,animals subjected to high doses of VX-745 experienced adverseneurological effects, although these adverse events were not observed inhumans. Despite validating the proof-of-concept for the inhibition ofp38 MAPK as a treatment for RA, VX-745 was discontinued due to thepotential for serious adverse events.

Another study utilizing VX-745 as a reference compound in an arthritismodel demonstrated that a 10 mg/kg dose of VX-745 was not as effectiveat inhibiting paw swelling as other compounds assayed. See Chopra etal., “Pharmacological profile of AW-814141, a novel, potent, selectiveand orally active inhibitor of p38 MAP kinase,” InternationalImmunopharmacology, 10: 467-473 (2010), the entirety of which isincorporated herein by reference.

In an osteoarthritis model, VX-745 showed statistically significantinhibition of knee degeneration compared to control animals whenadministered to rats at 50 mg/kg. VX-745 was also assayed in ahyperalgesia model and showed significant inhibition of hyperalgesicresponse when administered to rats at doses of 30 mg/kg, 10 mg/kg and 3mg/kg. The researchers discovered that the mice exhibited hyperalgesiaat the 3 mg/kg, 10 mg/kg and 30 mg/kg doses. However, the researchersobserved minimal effect at the 3 mg/kg dose. See Brown et al., “p38 MAPkinase inhibitors as potential therapeutics for the treatment of jointdegeneration and pain associated with osteoarthritis,” J. Inflamm., 5:22(2008), the entirety of which is incorporated herein by reference.Without wishing to be bound by theory, it is believed that the clinicalfailures of p38 inhibitors to treat chronic conditions such asrheumatoid arthritis are due to redundancy of the inflammatory pathway.Such redundancy results in the upregulation of feedback loops when p38is chronically inhibited, leading to an overall lack of efficacy.

Methods of the Invention

As described above, in some embodiments, the present invention providesa method of reducing amyloid plaque burden within the CNS. In someembodiments, a method of reducing amyloid plaque burden comprisesadministering to a patient in need thereof a p38 MAPK inhibitor.

In some embodiments, a method of reducing amyloid plaque burdencomprises administering to a patient in need thereof a selective p38MAPK inhibitor.

In certain embodiments, the present invention provides a method ofreducing amyloid plaque burden by administering to a patient in needthereof a low dose of a p38 MAPK inhibitor. In some embodiments, amethod of reducing amyloid plaque burden comprises administering to apatient in need thereof VX-745. In some such embodiments, a method ofreducing amyloid plaque burden comprises administering to a patient inneed thereof a low dose of VX-745.

An agent's therapeutic efficacy is affected by the degree to which itbinds blood plasma proteins. Only the fraction of unbound agent exhibitsany pharmacological effect because protein-bound agents cannot traversecell membranes or diffuse throughout the body. Thus, the more highlybound a therapeutic agent is, the lower the concentration of the agentavailable to elicit the desired pharmacological response. However,because there is less protein in the brain, a therapeutic agent which iscapable of crossing the blood-brain barrier will have a higherconcentration of free agent available to elicit the desiredpharmacological response. Indeed, although it is known that VX-745 is ahighly protein-bound agent, its brain levels in dogs is twice that ofsystemic levels.

In some embodiments, the present invention provides a method of reducingamyloid plaque burden associated with Alzheimer's disease comprisingadministering to a patient in need thereof a low dose of a p38 MAPKinhibitor. In some embodiments, the present invention provides a methodof reducing amyloid plaque burden associated with Alzheimer's diseasecomprising administering to a patient in need thereof VX-745. In someembodiments, the present invention provides a method of reducing amyloidplaque burden associated with Alzheimer's disease comprisingadministering to a patient in need thereof a low dose of VX-745.

In some embodiments, the present invention provides a method of (i)reducing plaque burden and (ii) inhibiting MAPT phosphorylation. In someembodiments, a method of (i) reducing plaque burden and (ii) inhibitingMAPT phosphorylation comprises administering to a patient in needthereof a p38 MAPK inhibitor. In certain embodiments, a method of (i)reducing plaque burden and (ii) inhibiting MAPT phosphorylationcomprises administering to a patient in need thereof a low dose of a p38MAPK inhibitor. In some embodiments, a method of (i) reducing plaqueburden and (ii) inhibiting MAPT phosphorylation comprises administeringto a patient in need thereof VX-745. In certain embodiments, a method of(i) reducing plaque burden and (ii) inhibiting MAPT phosphorylationcomprises administering to a patient in need thereof a low dose ofVX-745.

As discussed above, while IL-1β overexpression enhances Aβ clearance,chronic activation of microglia reduces the ability of microglia todegrade Aβ. Thus, in some embodiments, the present invention provides amethod of reducing plaque burden without inducing neuroinflammation. Insome embodiments, a method of reducing plaque burden without inducingneuroinflammation comprises administering to a patient in need thereof ap38 MAPK inhibitor. In some embodiments, a method of reducing plaqueburden without inducing neuroinflammation comprises administering to apatient in need thereof a low dose of a p38 MAPK inhibitor. In someembodiments, a method of reducing plaque burden without inducingneuroinflammation comprises administering to a patient in need thereofVX-745. In some embodiments, a method of reducing plaque burden withoutinducing neuroinflammation comprises administering to a patient in needthereof a low dose of VX-745.

In some embodiments, the present invention provides a method of reducingplaque burden without increasing expression and/or levels ofinflammatory cytokines. In some embodiments, a method of reducing plaqueburden without increasing expression and/or levels of inflammatorycytokines comprises administering to a patient in need thereof a p38MAPK inhibitor. In some embodiments, a method of reducing plaque burdenwithout increasing expression and/or levels of inflammatory cytokinescomprises administering to a patient in need thereof a low dose of a p38MAPK inhibitor. In some embodiments, a method of reducing plaque burdenwithout increasing expression and/or levels of inflammatory cytokinescomprises administering to a patient in need thereof VX-745. In someembodiments, a method of reducing plaque burden without increasingexpression and/or levels of inflammatory cytokines comprisesadministering to a patient in need thereof a low dose of VX-745.

In some embodiments, the present invention provides a method of reducingplaque burden without increasing IL-1β expression and/or levels. In someembodiments, a method of reducing plaque burden without increasing IL-1βexpression and/or levels comprises administering to a patient in needthereof a p38 MAPK inhibitor. In certain embodiments, the presentinvention provides a method of reducing plaque burden without increasingIL-1β expression and/or levels by administering to a patient in needthereof a low dose of a p38 MAPK inhibitor. In some embodiments, amethod of reducing plaque burden without increasing IL-1β expressionand/or levels comprises administering to a patient in need thereofVX-745. In certain embodiments, the present invention provides a methodof reducing plaque burden without increasing IL-1β expression and/orlevels by administering to a patient in need thereof a low dose ofVX-745.

In some embodiments, the present invention provides for theadministration of a p38 inhibitor, for example VX-745, once a day, twicea day, once a week, twice a week or once a month. In some embodiments,the present invention provides for the administration of a p38 inhibitorat more frequent intervals, such as one, two, three or four times perday, for up to one, two or three or more weeks, followed by a tapereddosing schedule to maintain the desired level of the p38 inhibitor. Insome embodiments, the present invention provides for the administrationof a p38 inhibitor at intervals of one, two, three or four times perday, for up to one, two or three or more months, followed by a tapereddosing schedule to maintain the desired level of the p38 inhibitor. Moreparticularly, in some embodiments, the present invention provides adosing schedule for the administration of a p38 inhibitor at intervalssufficient to achieve therapeutic levels in the brain, followed by atapering of the dosage.

In some embodiments, the present invention provides a method of reducingthe number and/or volume of amyloid plaques in a patient suffering fromAlzheimer's disease comprising administering to the patient a p38inhibitor. In some embodiments, the present invention provides a methodof reducing the number and/or volume of amyloid plaques in a patientsuffering from Alzheimer's disease comprising administering to thepatient a low dose of a p38 inhibitor. In some embodiments, the presentinvention provides a method of reducing the number and/or volume ofamyloid plaques in a patient suffering from Alzheimer's diseasecomprising administering to the patient a therapeutically effective doseof VX-745. In some embodiments, the present invention provides a methodof reducing the number and/or volume of amyloid plaques in a patientsuffering from Alzheimer's disease comprising administering to thepatient a low dose of VX-745.

In some embodiments, the present invention provides a method of reducingthe number and/or volume of amyloid plaque Aβ₄₂ in a patient sufferingfrom Alzheimer's disease comprising administering to the patient a p38inhibitor. In some embodiments, the present invention provides a methodof reducing the number and/or volume of amyloid plaque Aβ₄₂ in a patientsuffering from Alzheimer's disease comprising administering to thepatient a low dose of a p38 inhibitor. In some embodiments, the presentinvention provides a method of reducing the number and/or volume ofamyloid plaque Aβ₄₂ in a patient suffering from Alzheimer's diseasecomprising administering to the patient VX-745. In some embodiments, thepresent invention provides a method of reducing the number and/or volumeof amyloid plaque Aβ₄₂ in a patient suffering from Alzheimer's diseasecomprising administering to the patient a low dose of VX-745.

In some embodiments, the present invention provides a method of reducingthe number and/or volume of a beta amyloid plaque in a patient sufferingfrom Alzheimer's disease comprising administering to the patient atherapeutically effective dose of a p38 inhibitor. In some embodiments,the present invention provides a method of reducing the number and/orvolume of a beta amyloid plaque in a patient suffering from Alzheimer'sdisease comprising administering to the patient a therapeuticallyeffective dose of VX-745. In some embodiments, the present inventionprovides a method of reducing the number and/or volume of amyloid plaqueAβ₄₂ in a patient suffering from Alzheimer's disease comprisingadministering to the patient a low dose of a p38 inhibitor. In someembodiments, the present invention provides a method of reducing thenumber and/or volume of amyloid plaque Aβ₄₂ in a patient suffering fromAlzheimer's disease comprising administering to the patient a low doseof VX-745.

In some embodiments, the present invention provides a method forpreventing the accumulation of amyloid plaques comprising theadministration to a patient in need thereof a therapeutically effectiveamount of a p38 inhibitor. In some embodiments, the present inventionprovides a method for preventing the accumulation of amyloid plaquescomprising the administration to a patient in need thereof atherapeutically effective amount of a low dose of a p38 inhibitor. Insome embodiments, the present invention provides a method for preventingthe accumulation of amyloid plaques comprising the administration to apatient in need thereof a therapeutically effective amount of VX-745. Insome embodiments, the present invention provides a method for preventingthe accumulation of amyloid plaques comprising the administration to apatient in need thereof a therapeutically effective amount of a low doseof VX-745.

In some embodiments, the present invention provides a method of reducingthe number and/or volume of amyloid plaques in a patient suffering fromAlzheimer's disease comprising administering to the patient atherapeutically effective dose of a p38 inhibitor, wherein thetherapeutically effective dose is between about 1 mg to about 50 mg. Insome embodiments, the present invention provides a method of reducingthe number and/or volume of amyloid plaques in a patient suffering fromAlzheimer's disease comprising administering to the patient atherapeutically effective dose of a p38 inhibitor, wherein thetherapeutically effective dose is between about 1 mg to about 20 mg. Insome embodiments, the present invention provides a method of reducingthe number and/or volume of amyloid plaques in a patient suffering fromAlzheimer's disease comprising administering to the patient atherapeutically effective dose of a p38 inhibitor, wherein thetherapeutically effective dose is between about 1 mg to about 10 mg. Insome embodiments, the present invention provides a method of reducingthe number and/or volume of amyloid plaques in a patient suffering fromAlzheimer's disease comprising administering to the patient atherapeutically effective dose of a p38 inhibitor, wherein thetherapeutically effective dose is between about 1 mg to about 5 mg. Insome embodiments, the present invention provides a method of reducingthe number and/or volume of amyloid plaques in a patient suffering fromAlzheimer's disease comprising administering to the patient atherapeutically effective dose of a p38 inhibitor, wherein thetherapeutically effective dose is between about 5 mg to about 10 mg. Insome embodiments, the present invention provides a method of reducingthe number and/or volume of amyloid plaques in a patient suffering fromAlzheimer's disease comprising administering to the patient atherapeutically effective dose of a p38 inhibitor, wherein thetherapeutically effective dose is between about 10 mg to about 20 mg. Insome embodiments, the present invention provides a method of reducingthe number and/or volume of amyloid plaques in a patient suffering fromAlzheimer's disease comprising administering to the patient atherapeutically effective dose of a p38 inhibitor, wherein thetherapeutically effective dose is between about 20 mg to about 30 mg. Insome embodiments, the present invention provides a method of reducingthe number and/or volume of amyloid plaques in a patient suffering fromAlzheimer's disease comprising administering to the patient atherapeutically effective dose of a p38 inhibitor, wherein thetherapeutically effective dose is between about 30 mg to about 40 mg. Insome embodiments, the present invention provides a method of reducingthe number and/or volume of amyloid plaques in a patient suffering fromAlzheimer's disease comprising administering to the patient atherapeutically effective dose of a p38 inhibitor, wherein thetherapeutically effective dose is between about 40 mg to about 50 mg.

In some embodiments, the present invention provides an amyloid plaqueclearance mechanism comprising administering to a subject in needthereof a p38 MAPK inhibitor.

In some embodiments, the present invention provides a method of reducingplaque burden in a patient in need thereof, said method comprisingadministering to said patient a p38 MAPK inhibitor for a period of lessthan about 6 months. In some embodiments, the present invention providesa method of reducing plaque burden in a patient in need thereof, saidmethod comprising administering to said patient a p38 MAPK inhibitor fora period of less than about 4 months. In some embodiments, the presentinvention provides a method of reducing plaque burden in a patient inneed thereof, said method comprising administering to said patient a p38MAPK inhibitor for a period of less than about 2 months. In someembodiments, the present invention provides a method of reducing plaqueburden in a patient in need thereof, said method comprisingadministering to said patient a p38 MAPK inhibitor for a period of lessthan about 1 month. In some embodiments, the present invention providesa method of reducing plaque burden in a patient in need thereof, saidmethod comprising administering to said patient a p38 MAPK inhibitor fora period of less than about 2 weeks.

In some embodiments, the present invention provides a method of reducingamyloid plaque burden, said method comprising:

-   -   (i) imaging the brain of a subject to produce a neuroimage;    -   (ii) comparing the neuroimage to a reference image to determine        the number and/or area of the amyloid plaques; and    -   (iii) administering a therapeutically effective amount of a p38        inhibitor if the subject is determined to have an increased        amount of amyloid plaques when compared to the reference image.

In some embodiments, the present invention provides a method of reducingamyloid plaque burden, said method comprising:

-   -   (i) imaging the brain of a subject;    -   (ii) determining the number and/or area of the amyloid plaques;        and    -   (iii) administering a therapeutically effective amount of a p38        inhibitor if the number and/or area of the amyloid plaques        exceeds a predetermined threshold.

In some embodiments, the reference image is an image of a controlsubject. In some embodiments, the reference image is an image of asubject having normal cognitive function. In some embodiments, thereference image is a baseline image of the subject's brain. In some suchembodiments, the reference image is a prior scan of the subject's brain.In some embodiments, the subject is at risk for developing Alzheimer'sdisease.

In some embodiments, steps of (i) imaging, (ii) comparing amyloidplaques to a reference image and/or determining the number and/or areaof amyloid plaques and (iii) administering a p38 inhibitor are repeatedat one or more predetermined intervals. In some such embodiments, apredetermined interval is one month, two months, three months, fourmonths, five months, six months, seven months, eight months, ninemonths, ten months, eleven months, twelve months. In some embodiments, apredetermined interval is one year, two years, three years, four yearsor five years. In some embodiments, the predetermined interval is six(6) months.

In some embodiments, the subject is a patient at risk of developing orsuffering from Alzheimer's disease.

In some embodiments, the brain of a subject is imaged using one or moreneuroimaging techniques. In some embodiments, the neuroimaging techniqueis selected from the group consisting of computerized axial tomography(CAT or CT), single photon emission computed tomography (SPECT),positron emission tomography (PET), magnetic resonance imaging (MRI) orfunctional magnetic resonance imaging (fMRI). In some embodiments, theneuroimaging technique is computerized axial tomography (CAT or CT). Insome embodiments, the neuroimaging technique is positron emissiontomography (PET). In some such embodiments, the imaging agent used inthe PET scan is selected from amyvid or Pittsburgh compound B. In someembodiments, the neuroimaging technique is magnetic resonance imaging(MRI). In some embodiments, the neuroimaging technique is functionalmagnetic resonance imaging (fMRI).

A person of ordinary skill understands how to determine or measure thenumber and/or area of the amyloid plaques in a neuroimage. For example,see Zeman et al., “Diagnosis of Dementia Using Nuclear Medicine ImagingModalities,” Chapter 8, 12 Chapters on Nuclear Medicine,Gholamrezanezhad, Ed., 199-229 (Dec. 22, 2011) and Hsiao et al.,“Correlation of early-phase ¹⁸F-florbetapir (AV-45/Amyvid) PET images toFDG images: preliminary studies,” European Journal of Nuclear Medicineand Molecular Imaging, 39(4), 613-620 (2012), the entirety of each ofwhich is hereby incorporated by reference in its entirety.

In some embodiments, a p38 inhibitor is administered for a period ofless than six (6) months. In some embodiments, a p38 inhibitor isadministered for a period of less than four (4) months. In someembodiments, a p38 inhibitor is administered for a period of less thantwo (2) months. In some embodiments, a p38 inhibitor is administered fora period of less than one (1) month. In some embodiments, a p38inhibitor is administered for a period of less than three (3) weeks. Insome embodiments, a p38 inhibitor is administered for a period of lessthan two (2) weeks. In some embodiments, a p38 inhibitor is administeredfor a period of less than one (1) week.

In some embodiments, a predetermined threshold is a baseline for aparticular subject. For example, in some embodiments, the brain of asubject at risk for developing Alzheimer's disease is imaged and thenumber and/or area of amyloid plaques is determined and/or measured. Thenumber and/or area of the plaques is that subject's baseline orpredetermined threshold against which all later brain images arecompared.

In some embodiments, a predetermined threshold is based on the numberand/or area of amyloid plaques typically found in an Alzheimer'sdiseased brain. In some such embodiments, a predetermined threshold isan average of the number and/or area of amyloid plaques typically foundin an Alzheimer's diseased brain.

In some embodiments, the present invention provides a method of reducingamyloid plaque burden in a subject suffering from or at risk fordeveloping Alzheimer's disease, said method comprising:

-   -   (iv) imaging the brain of a subject;    -   (v) determining the number and/or area of the amyloid plaques;        and    -   (vi) administering a therapeutically effective amount of a p38        inhibitor if the number and/or area of the amyloid plaques        exceeds a predetermined threshold.

In some embodiments, the present invention provides a method of treatinga subject suffering from amyloid plaques, wherein the number and/or areaof the amyloid plaques exceeds a predetermined threshold, said methodcomprising administering to the subject a therapeutically effectiveamount of a p38 inhibitor.

In some embodiments, the present invention provides a method of treatinga subject suffering from amyloid plaques, wherein the number and/or areaof the amyloid plaques exceeds a predetermined threshold, said methodcomprising:

-   -   (i) administering a therapeutically effective amount of a p38        inhibitor for a period of less than six (6) months;    -   (ii) imaging the brain of the subject at regular intervals; and    -   (iii) administering a therapeutically effective amount of a p38        inhibitor if the number and/or area of the amyloid plaques        exceeds the previously measured amyloid plaque level.

In some such embodiments, the subject is administered a p38 inhibitorfor a period of less than four (4) months, less than two (2) months,less than one (1) month, or less than two (2) weeks.

In some embodiments, the present invention provides a method of treatinga subject suffering from amyloid plaques, wherein the number and/or areaof the amyloid plaques exceeds a predetermined threshold as measured byone or more neuroimaging techniques, said method comprisingadministering a therapeutically effective amount of a p38 inhibitor fora period of less than six (6) months. In some such embodiments, theneuroimaging technique is a PET scan. In some embodiments, the methodfurther comprises (i) imaging the brain of the subject at regularintervals; and (ii) administering a therapeutically effective amount ofa p38 inhibitor if the number and/or area of the amyloid plaques exceedsthe previously measured amyloid plaque level.

Combination Therapies

In certain embodiments, the present invention provides a method oftreating Alzheimer's disease comprising administering to a subject atherapeutically effective amount of a p38 inhibitor together with one ormore additional therapeutic agents. In some embodiments, the presentinvention provides a method of treating Alzheimer's disease comprisingadministering to a subject a therapeutically effective amount of a p38inhibitor together with one or more additional therapeutic agentsselected from cholinesterase inhibitors, N-methyl-D-aspartateantagonists, vitamin E, antidepressants, anxiolytics, antipsychotics,mood stabilizers and sleep aids.

Representative cholinesterase inhibitors include, without limitation,donepezil (Aricept®), rivastigmine (Exelon®), galantamine (Razadyne®)and tacrine (Cognex®).

Representative antidepressants include, without limitation, bupropion(Wellbutrin®), citalopram (Celexa®), fluoxetine (Prozac®), mirtazapine(Remeron®), paroxetine (Paxil®), sertraline (Zoloft®), trazodone(Desyrel®), venlafaxine (Effexor®), nortriptyline (Pamelor®) anddesipramine (Norpramine®).

Representative anxiolytics include, without limitation, lorazepam(Ativan®) and oxazepam (Serax®).

Representative antipsychotics include, without limitation, aripiprazole(Abilify®), clozapine (Clozaril®), haloperidol (Haldol®), olanzapine(Zyprexa®), quetiapine (Seroquel®), risperidone (Risperdal®) andziprasidone (Geodon®).

Representative mood stabilizers include, without limitation,carbamazepine (Tegretol®) and divalproex (Depakota®).

Representative sleep aids include, without limitation, zolpidem,zaleplon and chloral hydrate.

Representative N-methyl-D-aspartate antagonists include, withoutlimitation, memantine (Namenda®).

In some embodiments, the present invention provides a method of treatingAlzheimer's disease comprising administering to a subject atherapeutically effective amount of a p38 inhibitor together with one ormore additional therapeutic agents selected from the group consisting ofexenatide (Byetta®), varenicline, PF-04360365, rivastigmine, LY450139,ST101, bryostatin, EVP-6124, atomoxetine, HF0220, resveratrol,galantamine, PF-01913539, semagacestat, 3APS, immunoglobulin, dimebon,alpha-tocopherol, BAY85-8101, estrogen, progesterone, ACC-001, ginkobiloba, nicergoline, piracetam, NIC5-15, xaliproden (SR57746A),indomethacin, DMXB-A, LY2062430, 11-C PIB, bapineuzumab, etanercept,ramipril, interferon beta-1a, simvastatin, lipoic acid, fish oil,curcumin, PF-04447943, folate, vitamin B6, vitamin B12, leuprolide,INM-176, AH110690, tryptophan, SK-PC-B70M, BMS-708163, escitalopram,TRx0014, BAY94-9172, cerebrolysin, epigallocatechin-galate, SB-742457,lithium, rosiglitazone, divalproex, SAR110894D, PRX-03140, CX516(Ampalex), nicotinamide, rasagiline, AC-1202 (Ketasyn®), enduramide,neramexane, razadyne, NS 2330 (Tesofensine®), tamibarotene, acitretin,methylphenidate, mifepristone, ZT-1, AFFITOPE AD01, AFFITOPE AD02,GSK239512, GSK933776, SR57667B, PPI-1019, MPC-7869, AZD3480, PAZ-417,solanezumab, masitinib (AB1010), BAY1006578, docosahexaenoic acid,QS-21, MNI-558, reminyl retard, flutemetamol, estradiol,medroxyprogesterone, valproate, T-817MA, AZD1446, AAB-003 (PF-05236812),modafinil, raloxifene, atorvastatin, doxycycline, trazadone, sodiumoxybate, huperzine A, lutein, zeaxanthin, AC-3933, dextroamphetamine,EPAX 1050TG, SRA-333, MNI-168, CAD106, SGS742, NP031112, SSR180711C,GSI-953, prazosin, MEM 1003, AndroGel, AVE1625, cyclophosphamate,TC-5619-238, MK0249, lecozotan, circadin, MEM 3454, PPI-1019, UB 311,PF-04494700, ABT-089, LY451395, E2020, Rofecoxib, PF-03654746, EHT 0202etazolate, DCB-AD1, ONO-2506P0, EGb761®, gantenerumab, florbetapir,ELND005, prednisone, novasoy, ginseng, pioglitazone, caprylidene,ABT-288, ABT-384, nefiracetam, AQW051, Pitavastatin, naproxen sodium(Aleve®), lornoxicam, AN-1792, SR57667B, melatonin, SAM-531, MK0952,MK0677, IFN-alpha2A, BAY 94-9172, PYM50028, lecozotan SR, thalidomide,tramiprosate, FK962, IVIG, R05313534, bifeprunox, LNK-754, ELND005,NSA-789, ramelteon, Florbetaben, SRA-444, VP4896, celecoxib,hydrocodone, GSI-136, Zolpidem, MK3328, metformin, CTS21166, elontril,ibuprofen, posiphen tartrate, JNJ-39393406, testosterone, BRL-049653,BMS-708163, SAM-315, ketoconazole, fluconazole, warfarin, E2609,AZD0328, LY2886721, CHF 5074, E2212, acetaminophen, LY2811376, ABT-126,melatonin, GSK1034702, armodafinil, depakote, gemfibrozil, AL-108,levetiracetam, and quinacrine.

Pharmaceutical Compositions

In some embodiments, the present invention provides a pharmaceuticalcomposition comprising a p38 MAPK inhibitor together with one or moretherapeutic agents and a pharmaceutically acceptable carrier, adjuvant,or vehicle. In some embodiments, the present invention provides apharmaceutical composition comprising a low dose of a p38 MAPK inhibitortogether with one or more therapeutic agents and a pharmaceuticallyacceptable carrier, adjuvant, or vehicle. In some embodiments, thepresent invention provides a pharmaceutical composition for treatingAlzheimer's disease comprising a p38 inhibitor and one or morepharmaceutically acceptable excipients. In some embodiments, the presentinvention provides a pharmaceutical composition for treating Alzheimer'sdisease comprising a p38 inhibitor selected from VX-702, VX-745, BIRB796, TAK-715, SCIO 469, RWJ 67657, SB 681323, SB 242235, SB 203580,L-167307, RPR-203494, RPR-200765A, PD 169316, SB 200025, JX 401, CMPD1,SKF 86002, SX 011, SD 282, EO 1428, SD 169, SB 220025, SB 202190, SB239063, Org 48762-0, LY2228820, vinorelbine, PH-797804 and asiatic acid,and one or more pharmaceutically acceptable excipients. In some suchembodiments, a pharmaceutical composition for treating Alzheimer'sdisease comprises a low dose p38 inhibitor. In some embodiments, thepresent invention provides a pharmaceutical composition for treatingAlzheimer's disease comprising VX-745. In some such embodiments, apharmaceutical composition comprises a low dose of VX-745.

In some embodiments, the present invention provides a pharmaceuticalcomposition comprising VX-745 together with one or more therapeuticagents and a pharmaceutically acceptable carrier, adjuvant, or vehicle.In some embodiments, the present invention provides a pharmaceuticalcomposition comprising a low dose of VX-745, one or more therapeuticagents and a pharmaceutically acceptable carrier, adjuvant, or vehicle.In some embodiments, the present invention provides a pharmaceuticalcomposition comprising a low dose of VX-745, one or more therapeuticagents selected from donepezil (Aricept®), rivastigmine (Exelon®),galantamine (Razadyne®), tacrine (Cognex®), bupropion (Wellbutrin®),citalopram (Celexa®), fluoxetine (Prozac®), mirtazapine (Remeron®),paroxetine (Paxil®), sertraline (Zoloft®), trazodone (Desyrel®),venlafaxine (Effexor®), nortriptyline (Pamelor®), desipramine(Norpramine®), lorazepam (Ativan®), oxazepam (Serax®), aripiprazole(Abilify®), clozapine (Clozaril®), haloperidol (Haldol®), olanzapine(Zyprexa®), quetiapine (Seroquel®), risperidone (Risperdal®),ziprasidone (Geodon®), carbamazepine (Tegretol®), divalproex(Depakota®), zolpidem, zaleplon, chloral hydrate, memantine (Namenda®),exenatide (Byetta®), varenicline, PF-04360365, rivastigmine, LY450139,ST101, bryostatin, EVP-6124, atomoxetine, HF0220, resveratrol,galantamine, PF-01913539, semagacestat, 3APS, immunoglobulin, dimebon,alpha-tocopherol, BAY85-8101, estrogen, progesterone, ACC-001, ginkobiloba, nicergoline, piracetam, NIC5-15, xaliproden (SR57746A),indomethacin, DMXB-A, LY2062430, 11-C PIB, bapineuzumab, etanercept,ramipril, interferon beta-1a, simvastatin, lipoic acid, fish oil,curcumin, PF-04447943, folate, vitamin B6, vitamin B12, leuprolide,INM-176, AH110690, tryptophan, SK-PC-B70M, BMS-708163, escitalopram,TRx0014, BAY94-9172, cerebrolysin, epigallocatechin-galate, SB-742457,lithium, rosiglitazone, divalproex, SAR110894D, PRX-03140, CX516(Ampalex), nicotinamide, rasagiline, AC-1202 (Ketasyn®), enduramide,neramexane, razadyne, NS 2330 (Tesofensine®), tamibarotene, acitretin,methylphenidate, mifepristone, ZT-1, AFFITOPE AD01, AFFITOPE AD02,GSK239512, GSK933776, SR57667B, PPI-1019, MPC-7869, AZD3480, PAZ-417,solanezumab, masitinib (AB1010), BAY1006578, docosahexaenoic acid,QS-21, MNI-558, reminyl retard, flutemetamol, estradiol,medroxyprogesterone, valproate, T-817MA, AZD1446, AAB-003 (PF-05236812),modafinil, raloxifene, atorvastatin, doxycycline, trazadone, sodiumoxybate, huperzine A, lutein, zeaxanthin, AC-3933, dextroamphetamine,EPAX 1050TG, SRA-333, MNI-168, CAD106, SGS742, NP031112, SSR180711C,GSI-953, prazosin, MEM 1003, AndroGel, AVE1625, cyclophosphamate,TC-5619-238, MK0249, lecozotan, circadin, MEM 3454, PPI-1019, UB 311,PF-04494700, ABT-089, LY451395, E2020, Rofecoxib, PF-03654746, EHT 0202etazolate, DCB-AD1, ONO-2506P0, EGb761®, gantenerumab, florbetapir,ELND005, prednisone, novasoy, ginseng, pioglitazone, caprylidene,ABT-288, ABT-384, nefiracetam, AQW051, Pitavastatin, naproxen sodium(Aleve®), lornoxicam, AN-1792, SR57667B, melatonin, SAM-531, MK0952,MK0677, IFN-alpha2A, BAY 94-9172, PYM50028, lecozotan SR, thalidomide,tramiprosate, FK962, IVIG, R05313534, bifeprunox, LNK-754, ELND005,NSA-789, ramelteon, Florbetaben, SRA-444, VP4896, celecoxib,hydrocodone, GSI-136, Zolpidem, MK3328, metformin, CTS21166, elontril,ibuprofen, posiphen tartrate, JNJ-39393406, testosterone, BRL-049653,BMS-708163, SAM-315, ketoconazole, fluconazole, warfarin, E2609,AZD0328, LY2886721, CHF 5074, E2212, acetaminophen, LY2811376, ABT-126,melatonin, GSK1034702, armodafinil, depakote, gemfibrozil, AL-108,levetiracetam, and quinacrine, and a pharmaceutically acceptablecarrier, adjuvant, or vehicle.

In certain embodiments, pharmaceutically acceptable compositions of thisinvention are formulated for oral administration. Pharmaceuticallyacceptable compositions of this invention may be orally administered inany orally acceptable dosage form including, but not limited to,capsules, caplets, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers commonly used include lactose andcorn starch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

The quantities of the compounds of the present invention that arecombined with the carrier materials to produce a composition in a singledosage form will vary depending upon the patient and the particular modeof administration. Preferably, provided compositions should beformulated so that a dosage of between 1-50 mg/day of the p38 inhibitor(ie, VX-745 or other p38 inhibitor) can be administered to a patientreceiving these compositions. Examples of compositions includecompositions formulated to administer dosages of between 1-10 mg, 10-25mg or 25-50 mg per day of the p38 inhibitor to the patient receivingthese compositions. In other embodiments of the invention, compositionsinclude compositions formulated to administer dosages of between 3-5 mg,5-10 mg, 10-20 mg, 20-30 mg, 30-40 mg or 40-50 mg, per day of theinhibitor to the patient receiving these compositions. In someembodiments, the composition is formulated into doses containing 1 mg, 3mg, 5 mg, 10 mg, 20 mg, 25 mg, 30 mg or 50 mg of the active composition.Dosing regimens for these formulations may include but are not limitedto single administration dosing, once, twice, or three times dailydosing, weekly dosing, and monthly dosing.

In some treatment regimens, patients will be initially treated withlarger doses of the compounds of the present invention (“loading dose”)for a certain period of time (“loading period”) in order to achieve ahigh tissue concentration of the drug, before being treated with lowerdoses of active composition (“maintenance dose”) for a longer period oftime (“maintenance period”) in order to maintain the serum or tissueconcentration of the active composition.

In some treatment regimens, administration of the inhibitor to a patientis temporarily halted (a “drug holiday”). In some examples, a patientmay have cycles of daily doses of inhibitor for a month followed by aone month holiday. In another example, a patient might have daily dosingof an inhibitor for six months, followed by a one month holiday. Inanother example, a patient might have daily doses of an inhibitor forthree weeks followed by a one week holiday. In yet another example, apatient might have daily doses of a drug for one week, followed by athree week holiday. In another example, a patient might have cycles ofweekly doses of a drug for 6 weeks, followed by a three week holiday.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of a compound of the present invention in the composition willalso depend upon the particular compound in the composition.

EXEMPLIFICATION Example 1

The purpose of the study was to evaluate the effect of 2 week twice aday oral VX-745 treatment on beta amyloid (Aβ) accumulation and plaqueload and inflammation in Alzheimer's disease (AD) transgenic Tg2576mouse model.

Animals. Transgenic mice were treated either with vehicle or VX-745 for2 weeks starting at 26 months of age. After 2 weeks of treatment theanimals were terminated, and the brains were used for biochemical andimmunohistological analyses for insoluble amyloid beta levels and plaqueload by Aβ1-42 ELISA and Aβ immunohistochemistry. Inflammation wasanalyzed by ventral cortex IL-1β and TNF-α ELISA and microgliosis byCD11b immunohistochemistry. Plasma was collected at end-point and sentto client for PK analysis.

All animal experiments were carried out according to the NationalInstitute of Health (NIH) guidelines for the care and use of laboratoryanimals, and approved by the State Provincial Office of SouthernFinland. Female transgenic Tg2576 mice (n=12) and wild-type mice (n=5),purchased from Taconic, were used for the experiment. Animals werehoused at a standard temperature (22±1° C.) and in a light-controlledenvironment (lights on from 7 am to 8 pm) with ad libitum access to foodand water. The Tg2576 transgenic line was developed through insertion ofthe hAPP695 construct with the ‘Swedish’ double mutation and hamsterprion protein cosmid vector into a C57B6/J×SJL host; the prion promoterlimits overexpression of mutant APP to neurons in the brain.Consequently, the Tg2576 mouse develops elevated brain levels of solubleAβ1-40 and Aβ1-42 by 6-8 months of age and Aβ-containing neuriticplaques in the neocortex and hippocampus by 10-16 months. The mice weredivided into treatment groups as follows:

-   -   5 wild-type control mice treated with Vehicle    -   6 Tg2576 mice treated with Vehicle    -   6 Tg2576 mice treated with VX-745 (3 mg/kg)

Compound Storage and Instructions for Formulation.

VX-745 was delivered to Cerebricon as dry compound by the sponsor. Thevehicle to be utilized was 1% Pluronic F108. The storage and dissolvinginstructions were provided by the sponsor. Material safety data sheet orsimilar document of the compound was provided by the sponsor. Thesolutions were stored according to instructions provided by the sponsor(storage conditions and expiration day of solution). Vehicle wasprovided by the sponsor/Cerebricon.

Drug Delivery.

The oral administration of VX-745 or vehicle by oral gavage (10 ml/kg)was done BID starting at the age of 26 months and continuing for 14days. On the day of termination, treatment was given 2 hours prior totermination.

General Health Status and Humane End-Points.

Animals were monitored twice-a-day by laboratory personnel (8 am and 4pm). In cases where general health status of an animal significantlyworsened, the mouse was terminated by an overdose of CO₂, decapitatedand brains processed as detailed below. Definitions of acceptableendpoints included: no spontaneous movements and inability to drink oreat in 24-h observation period, massive bleeding, spontaneousinflammation, missing anatomy, swelling or tumors.

Collection of Plasma and Brain Samples.

Two hours after the last dosing the mice were deeply anesthetized withsodium pentobarbital (60 mg/kg Mebunat, Orion Pharma, Finland). The micewere subjected to cardiac puncture and blood samples were collected intopre-cooled (ice bath) EDTA tubes. The tubes were kept on ice and plasmawas separated by centrifugation at 2000 g (+4° C.) as soon as possible.150-200 μl of plasma from each mouse was transferred into pre-cooledpolypropylene tubes and kept frozen at −80° C. until sent to the sponsorfor PK analysis.

The brains were perfused with non-heparinized saline. Right hemispherewas post-fixed by immersion in 4% PFA in 0.1 M PB. After a brief washwith phosphate buffer, it was cryoprotected in 30% sucrose in PB for 2-3days, after which it was frozen on liquid nitrogen and stored at −80° C.for further analysis (immunohistochemistry). Left hemisphere (dissectedon ice to hippocampus, ventral and dorsal cortex and the rest fractions)was fresh-frozen on dry ice and stored at −80° C. for biochemicalanalysis (ELISA). Cerebellum was fresh-frozen and stored at −80° C. foroptional future PK/other analysis.

Immunohistochemistry.

Twenty-μm-thick coronal sections were prepared with a cryostat andmounted on SuperFrost Plus glass slides from the fixed, cryoprotectedand frozen hemispheres. Selected sections were used forimmunohistochemical analyses. Plaque load and the degree of amyloidaggregates in cortical and hippocampal structures were analyzed withamyloid beta immunohistochemical staining.

From the adjacent sections, degree of microgliosis was analyzed withCD11b immunohistochemistry.

Aβ and CD11b Immunohistochemistry:

Briefly, tissue sections used in immunohistochemistry were thawed andair dried. After blocking the internal peroxidase activity andunspecific binding, and washes, sections were reacted overnight at RTwith:

-   -   anti-Aβ (mouse anti-Aβ[4-10], the Genetics Company AB02,        1:20,000, clone W0-2)    -   anti-CD11b (rat anti-CD11b, AbD Serotec Inc. MCA711, 1:500)

Thereafter the sections were incubated with proper biotinylatedsecondary antibody and avidin-biotin complex (Vectastain Elite kit,Vector Laboratories, Burlingame, Calif.) for 2 h each. The peroxidasecontaining avidin-biotin complex was visualized using nickel-enhancedDAB as a substrate. Finally, the sections were rinsed, dehydrated,coverslipped and examined with a Leica 3000RB microscope.

Image Analysis.

Equally spaced coronal tissue sections along the antero-posterior axisof the hippocampus (3-4 tissue sections from each animal) were analyzedfor immunostaining intensity by ImagePro Plus software. Images ofimmunoreactive staining were captured at defined light and filtersettings in a brightfield microscope equipped with a color CCD-camera.The captured images of Aβ-immunoreactive plaque deposits andintraneuronal Aβ aggregates as well as CD11b immunoreactive images wereconverted to grayscale images, processed with a delineation function tosharpen edges to allow an accurate segmentation. The images weresegmented with an auto-threshold command (ImageProPlus,MediaCybernetics). The results were expressed as area fraction (stainedarea_(tot)/measured area_(tot), expressed in %) and presented asmean±SEM among the tissue sections analyzed from each individualtransgenic mouse. Ventral cortex and dorsal hippocampus were analyzedfrom the coronal sections (at the AP level of dorsal hippocampus).

Insoluble and Soluble Amyloid Beta 1-42 ELISA. Amyloid beta 1-42 ELISAanalyses were applied to detect insoluble and soluble form of Aβ₁₋₄₂ inventral cortex.

The ventral cortex tissue sample was homogenized and samples preparedaccording to the manufacturers detailed instructions (the GeneticsCompany, Switzerland, hAmyloid B42 Brain ELISA). Briefly the tissueswere homogenized with a Dounce homogenizer (2×10 strokes on ice) inlysis buffer at a ratio of 1:10 (tissue weight:lysis buffer). Lysisbuffer was Tris-buffered saline (TBS; 20 mM Tris-base and 137 mM NaCl,pH7.4) with protease inhibitors. The homogenate was centrifuged for 10min at +4° C. with 13,000 rpm and the supernatant was divided inaliqouts and stored frozen at −20° C. prior to analyses (=Soluble AB).

The pellet was re-homogenized in cold 70% formic acid in distilledwater, sonicated for 10 min, neutralized with 15× volume 1M Tris pH 7.4,and centrifuged for 10 min at +4° C. with 13,000 rpm. The supernatantwas stored frozen at −20° C. (=Insoluble AB).

Aβ₁₋₄₂ levels in insoluble and soluble fractions of brain tissue sampleswere analyzed with ELISA using Amyloid Beta 1-42 ELISA kits (hAmyloidB42 Brain ELISAs, The Genetics Company, Switzerland) according toinstructions of the manufacturer. Standard curve range was from 25 to500 pg/ml. FIGS. 1A and 1B depict the area percentage of Aβ₁₋₄₂ amyloidplaques of transgenic mice following a two-week administration of VX-7453 mg/kg BID. Of particular note, the present study was conducted onolder mice (26 months of age). Other studies attempt to prevent amyloidplaque accumulation and are thus conducted on mice of about 4 and/or 8months of age (see, for example, Zhu et al., J. Neuroscience, 31(4):1355-136 (2011), incorporated herein by reference in its entirety).Tg2576 mice aged 26 months have elevated brain levels of soluble amyloidplaque by 6-8 months of age. The present experiments were designed toevaluate the amyloid plaque clearing ability of a p38 inhibitor (i.e.,VX-745). Significantly, VX-745 showed a 32.5% decrease of amyloid plaquearea in the cortex as compared with vehicle (mean 27.7% amyloid plaquearea in control vs. mean 18.7% amyloid plaque area in VX-745-treatedanimals). VX-745 showed a 61.8% decrease of amyloid plaque area in thehippocampus as compared with vehicle (mean 13.6% amyloid plaque area incontrol vs. mean 5.2% amyloid plaque area in VX-745-treated animals).

IL-1β and TNF-α ELISA. IL-1β and TNF-α levels were analyzed from thesoluble dorsal cortex brain tissue fraction with mouse IL-1β and TNF-αELISA Kits (Quantikine M Cytokine mouse IL-1β and TNF-α ELISA kits,RND-Systems, MLB00 and MTA00, R&D Systems, MN, USA) according toinstructions of the manufacturer. FIG. 2 depicts the IL-1β levels intransgenic mice following a two-week administration of VX-745 3 mg/kgBID. Increases in inflammation, particularly neuroinflammation, areknown to trigger MAPT phosphorylation and aggregation throughoverexpression of IL-1. FIG. 2 depicts the IL-1β levels in treated micevs. control and wild type mice. Notably, the VX-745-treated mice showedno increases in IL-1β levels when compared to the wild-type or controlanimals.

Statistical Analysis.

All data were presented as mean±standard deviation (SD) or standarderror of mean (SEM), and differences were considered to be statisticallysignificant at the P<0.05 level. Statistical analysis was performedusing StatsDirect statistical software. Differences between group meanswere analyzed by using un-paired t-test.

1-3. (canceled)
 4. A method of treating a patient suffering fromAlzheimer's Disease, the method comprising administering to the patienta pharmaceutical composition comprising VX-745.
 5. The method of claim4, wherein the patient has brain amyloid plaques.
 6. The method of claim4, wherein the pharmaceutical composition comprises VX-745 at a unitdose of less than 250 mg.
 7. The method of claim 4, wherein thepharmaceutical composition is administered to the patient twice per day.8. The method of claim 4, wherein the pharmaceutical composition isadministered to the patient once per day.
 9. The method of claim 4,wherein the pharmaceutical composition is formulated for oraladministration.